The objective of the proposed research is to determine the mechanism, molecular properties and mode of regulation of nitrate reductase. We will test a model for the regulation of nitrate reductase which proposes that the processes of nitrate assimilation and CO2 fixation are coordinately regulated by the intracellular ratio of (O2)/(CO2) and that cyanide is a physiological regulator of nitrate reductase activity. The quaternary structure of nitrate reductase will be determined through end group analyses and ultracentrifugal analyses. The functions of the prosthetic groups and arrangements on the subunits will be studied by limited proteolysis, detergent treatment and site-specific reagents combined with analyses by gel electrophoresis, gel chromatography and gel isoelectric focusing. The reaction mechanism for nitrate reductase will be studied by steady state kinetics and rapid mixing-rapid freeze EPR techniques. EPR studies will also provide further information on the Mo-center of nitrate reductase and the mechanism of inactivation by cyanide. The intracellular location of the enzyme will be determined by immunofluorescence techniques in combination with electron microscopy and density gradient fractionation.